Initiation is the most complex stage of eukaryotic protein synthesis, and is a target for multiple regulatory pathways. Defects in the translation apparatus and its regulation cause a variety of devastating diseases, and initiation is therefore increasingly becoming a focus for chemo-therapeutic intervention. Knowledge of molecular mechanisms underlying initiation is thus an important priority, but details of several steps in thi process remain poorly understood. In outline, eukaryotic initiation occurs as follows. Met-tRNAiMet, eIF2 and GTP form a ternary complex, which together with eIFs 3, 1, and 1A bind to 40S subunits, yielding a 43S preinitiation complex. 43S complexes attach to the cap-proximal region of mRNA in a step that involves unwinding of its secondary structure by eIFs 4A, 4B and 4F, and then scan to the initiation codon, where they form 48S initiation complexes with established codon-anticodon base-pairing. Scanning on structured mRNAs strictly requires DHX29, a DExH-box protein that binds directly to 40S subunits. Subsequent joining of 60S subunits to 48S complexes and formation of elongation-competent 80S ribosomes is promoted by eIFs 5 and 5B. Our development of in vitro reconstitution of the entire translation process and recent integration of these approaches with advances in cryo-electron microscopy achieved by our collaborator J. Frank (HHMI, Columbia University) gives unique opportunities to close several critical gaps in understanding of the mechanism of mammalian initiation. Aims 1-3 of this proposal will address the key unresolved aspects of the first stages in the canonical initiation process: ribosomal recruitment of Met-tRNAiMet, attachment of 43S complexes to mRNA and ribosomal scanning. Aim 1 will focus on the mechanism by which eIF2 promotes binding of Met-tRNAiMet to the 40S subunit, in particular, (i) the role in this process of the eIF2 subunit, which was recently found to mediate the only stable contact of eIF2 with the 40S subunit in 43S complexes, and (ii) the mechanism by which the activity of eIF2 is stimulated by ABC50. In Aim 2, we will continue to further investigate the mechanistic aspects of entry of eIF4E-bound capped mRNAs into the mRNA-binding cleft of the 40S subunit during attachment of 43S complexes, and will characterize the role of eIF4B in this process. Aim 3 will be devoted to determination of the mechanism by which DHX29 promotes ribosomal scanning, in particular, to discrimination between direct unwinding of mRNA by DHX29 and an indirect mode of DHX29's action by inducing conformational changes in the 40S subunit. The last Aim 4 will concern non-canonical modes of initiation on non-AUG codons: (i) initiation on CUG codons with Leu-tRNALeu, which has the potential to be a regulatory mechanism allowing translation in conditions of eIF2 phosphorylation, and (ii) initiation on non-AUG triplets during repeat-associated non-AUG (RAN) translation, which occurs on triplet/sextet expansion repeats in noncoding regions of mRNAs transcribed from genes that are responsible for many severe neurodegenerative diseases.